Substantia nigra dopamine (SNc) neurons fire tonically at low rates resulting in action potential backpropagation. In a recently completed study from our lab, we found that the rate of background tonic firing exerts a powerful influence over dendritically-located conductances and synaptically-driven firing responses. Using two-photon glutamate-uncaging and synaptic stimulation, we show that fast tonic firing (4-5 Hz) intensifies synaptically-driven burst firing output in an NMDA-receptor dependent manner. Furthermore, we found that increasing the tonic rate from 1 to 6 Hz dramatically enhanced the average dendritic Ca signals, with some cells showing up to five-fold increase over linearly predicted Ca values. By contrast to SNc neurons, ventral tegmental area (VTA) dopamine neurons showed much more linear increases in Ca with rate suggesting a higher load of free Ca in SNc neurons. This study was submitted and has been published (Hage and Khaliq, 2015). Continuing our interest in examining functional heterogeneity among midbrain dopamine neurons, we have two projects nearing completion that identify important differences in subthreshold conductances that shape firing and dendritic excitability in subpopulations of dopamine neurons. In the first study, we compared retrogradely-labeled dopamine neurons that project to prefrontal cortex, nucleus accumbens and dorsal striatum. By comparison to SNc subpopulations (nigrostriatal neurons), we find that VTA subpopulations (mesocortical and mesoaccumbal neurons) show stronger sensitivity and longer delays to spike onset following hyperpolarizing stimuli. This differential sensitivity is due to the presence of a slowly decaying potassium current that is more strongly expressed in VTA dopamine neurons. Our plan is to test the implications of this finding on spike timing and synchrony among dopamine neuron subpopulations. In a second study, we examine dendritic excitability in SNc dopamine neurons. In past electrophysiological studies, SNc neurons were considered a much more homogeneous population than VTA neurons. Studying dendritic Ca signals of these cells, however, we have discovered dramatic differences in the strength and intensity of subthreshold responses in among SNc neurons. In particular, we find that a major fraction of SNc dopamine neurons exhibits non-linear depolarizing burst-like responses to evoked and synaptic inputs. These non-linear responses are driven by dendritic T-type Ca channels, and surprisingly, tend to be present almost exclusively in calbindin-negative SNc dopaminergic neurons. This finding has potential clinical relevance because calbindin-negative dopamine neurons tend to selectively die in Parkinsons patients. Along with our recently published results (Hage and Khaliq, 2015), this study supports the idea that Ca conductances play a much more dominant role in shaping the physiology of SNc dopaminergic neurons. Following our interest in the synaptic physiology of dopamine neurons, we have been examining the properties of dendritic spines on dopamine neurons. Anatomical evidence for the presence of dendritic spines has been mixed with some studies observing the presence of spine-like processes while others conclude that dopamine neurons are largely aspiny. We find that dopamine neurons clearly express spines in both juvenile and adult mice. We have confirmed that spines are present in fast perfused tissue preparations as well as golgi-stained tissue. Furthermore, we have tested the functionality using a combination of Ca imaging in the spine head and glutamate uncaging. These experiments show that they indeed express synaptic receptors and likely form functional synapses. We examined the effect of spine length on the function of spines and are currently comparing integration onto spines versus onto dendritic shaft synapses. This project is nearly completed and we plan to submit in the near future. Lastly, we participated in a collaborative project with the laboratory of Dr. Ellen Sidransky. A manuscript of this study was recently submitted and is under review.